Abstract Clathrin-mediated endocytosis is an evolutionarily ancient and fundamental membrane sorting process. It serves as a conduit to convey (sometimes temporarily) macromolecular cargo from the cell surface for delivery to intracellular organelles within small, membrane-encapsulated transport vehicles. Defects in clathrin- mediated endocytosis are linked to several important human disease states, from hypercholesterolemia and certain cancers to Alzheimer disease. Because endocytic clathrin coats are manufactured only to ferry designated cargo into the cytosol, and because the assembly process fails if cargo loading is defective, understanding the dynamics and regulation of cargo molecule recognition and capture is at the very center of a clearer mechanistic model of this vital process. Certainly, the complexity of cargoes that need to be differentially sorted into the cell is vast, and various, structurally discrete and non-competitive sorting signals operate to fill coated vesicles for each budding event. We have been investigating how this broad array of cargo is recognized, and how this is integrated into the polymeric clathrin coat assembly process that begins the construction of a nascent coat. The guiding central hypothesis is that in addition to the central sorting adaptor, the heterotetrameric AP-2 complex, versatile cargo recognition proteins expand the packaging repertoire. During the past funding period, a significant advance was the use of the zebrafish animal model to show the interrelated operation of AP-2 and one particular clathrin-associated sorting protein (CLASP), designated Fcho1, during early embryonic development. On the basis of discrete embryonic phenotypes following gene silencing, we identified that Fcho1 is necessary for proper bone morphogenetic protein (Bmp) signaling that governs ventral cell fates and axis patterning. By contrast, AP-2 gene silencing leads to a multifaceted, severe and penetrant embryonic failure. The overarching goal of the current application is to build on these findings and delineate how the functional surfaces on AP-2 and Fcho1 permit associations with the requisite binding partners. Through integrated structure?function experiments utilizing biochemical studies, cellular trafficking assays, cell-based imaging and whole animal genetic approaches, we will establish the molecular basis for the operation of these sorting adaptors during development and differentiation. In particular, we will test whether reversible phosphorylation of Tyr888 in the AP-2 ?2 subunit turns CLASP binding on and off, how AP-2 promotes receptor trafficking and signaling during early embryogenesis, investigate the molecular basis for the Fcho1 -homology domain binding to various partner proteins, and test whether Fcho1-dependent delivery of the Bmp receptor Alk8 is necessary for endosomal propagation of R- Smad signaling during embryogenesis. The new information generated is likely to provide novel mechanistic insights and important conceptual advances in the appreciation of clathrin-mediated endocytosis.